Viral particle-based protein-protein interaction

ABSTRACT

The disclosure relates to a virus-like particle in which a protein complex is entrapped, ensuring the formation of the protein complex under physiological conditions, while protecting the protein complex during purification and identification. The disclosure further relates to the use of such virus-like particle for the isolation and identification of protein complexes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national phase entry under 35 U.S.C. §371 ofInternational Patent Application PCT/EP2013/060787, filed May 24, 2013,designating the United States of America and published in English asInternational Patent Publication WO 2013/174999 A1 on Nov. 28, 2013,which claims the benefit under Article 8 of the Patent CooperationTreaty to European Patent Application Serial No. 12169209.9, filed May24, 2012.

TECHNICAL FIELD

The disclosure relates generally to biotechnology, and more particularlyto a virus-like particle in which a protein complex is entrapped,ensuring the formation of the protein complex under physiologicalconditions, while protecting the protein complex during purification andidentification. The disclosure further relates to the use of suchvirus-like particle for the isolation and identification of proteincomplexes.

BACKGROUND

Protein-protein interactions are an essential key in all biologicalprocesses, from the replication and expression of genes, to themorphogenesis of organisms. Protein-protein interactions govern, amongstothers, ligand-receptor interaction and the subsequent signalingpathway; they are important in assembly of enzyme subunits, in theformation of biological supramolecular structures such as ribosomes,filaments and virus particles, and in antigen-antibody interactions.

Researchers have developed several approaches in attempts to identifyprotein-protein interactions. A major breakthrough was obtained by theintroduction of the genetic approaches, of which the yeast two-hybrid(Fields and Song, 1989) is the most important one. Although thistechnique became widely used, it has several drawbacks. The fusionproteins need to be translocated to the nucleus, which is not alwaysevident. Proteins with intrinsic transcription activation properties maycause false positives. Moreover, interactions that are dependent uponsecondary modifications of the protein such as phosphorylation cannot beeasily detected.

Several alternative systems have been developed to solve one or more ofthese problems.

Approaches based on phage display do avoid nuclear translocation.WO9002809 describes how a binding protein can be displayed on thesurface of a genetic package, such as a filamentous phage, wherein thegene encoding the binding protein is packaged inside the phage. Phagesthat bear the binding protein that recognizes the target molecule areisolated and amplified. Several improvements of the phage displayapproach have been proposed, as described, e.g., in WO9220791,WO9710330, and WO9732017.

However, all these methods suffer from the difficulties that areinherent at the phage display methodology: the proteins need to beexposed at the phage surface and are so exposed to an environment thatis not physiologically relevant for the in vivo interaction. Moreover,when screening a phage library, there will be a competition between thephages that results in a selection of the high-affinity binders.

U.S. Pat. No. 5,637,463 describes an improvement of the yeast two-hybridsystem, whereby it can be screened for modification-dependentprotein-protein interactions. However, this method relies on theco-expression of the modifying enzyme, which will exert its activity inthe cytoplasm and may modify enzymes other than the one involved in theprotein-protein interaction, which may, on its turn, affect theviability of the host organism.

An interesting evolution is described in U.S. Pat. No. 5,776,689, by theso-called protein recruitment system. Protein-protein interactions aredetected by recruitment of a guanine nucleotide exchange factor (Sos) tothe plasma membrane, where Sos activates a Ras reporter molecule. Thisresults in the survival of the cell that otherwise would not survive inthe culture conditions used. Although this method has certainly theadvantage that the protein-protein interaction takes place underphysiological conditions in the submembrane space, it has severaldrawbacks. Modification-dependent interactions cannot be detected.Moreover, the method is using the pleiotropic Ras pathway, which maycause technical complications, such as the occurrence of falsepositives.

A major improvement in the detection of protein-protein interactions wasdisclosed in WO0190188, describing the so-called Mappit system. Themethod, based on a cytokine receptor, not only allows a reliabledetection of protein-protein interactions in mammalian cells, but alsomodification-dependent protein interactions can be detected, as well ascomplex three-hybrid protein-protein interactions mediated by a smallcompound (Caligiuri et al., 2006). However, although very useful, thesystem is limited in sensitivity and some weak interactions cannot bedetected. Moreover, as this is a membrane-based system, nuclearinteractions are normally not detected. Recently, a cytoplasmicinteraction trap has been described, solving several of thoseshortcomings. However, all of these “genetic” systems rely on theoverexpression of both interaction partners, which may result in falsepositives due to the artificial increase in concentration of one or bothof the interaction partners.

As an alternative for the genetic protein-protein interaction detectionmethods described above, biochemical or co-purification strategies,combined with mass spectrometry-based proteomics (Paul et al., 2011;Gingras et al., 2007), can be used. For the co-purification strategies,a cell homogenate is typically prepared by a detergent-based lysisprotocol, followed by capture using a (dual) tag approach (Gavin et al.,2002) or via specific antibodies (Malovannaya et al.). The proteincomplex extracted from the “soup” of cell constituents is then expectedto survive several washing steps, mostly to compensate for thesensitivity of contemporary MS instruments, before the actual analysisoccurs. There are no clear guidelines on the extent of washing or onavailable buffers and their stringency. Most lysis and washing protocolsare purely empirical in nature and were optimized using modelinteractions. It is, therefore, hard to speculate on the loss of factorsduring these steps (false negatives), or the possibility of falseinteractions due to loss of cellular integrity (false positives). Use ofmetabolic labeling strategies allows separation between the proteinssticking to the purification matrix, and between the proteins thatassociate specifically to the bait protein. Depending on thepurification conditions and the sensitivity of the MS instruments used,it is no exception to find more than 1000 proteins in the elutedfraction of a gel-free AP-MS experiment.

There is a further need for co-purification techniques, isolating theprotein complexes in their physiological environment, but wherein thecomplex is protected during the further purification and analysis.

The evolutionary stress on viruses promotes highly condensed coding ofinformation and maximal functionality for small genomes. Accordingly,for HIV-1, it is sufficient to express a single viral protein, the p55GAG protein, to allow the efficient production of virus-like particles(VLPs) from cells (Gheysen et al., 1989; Shioda and Shibuta, 1990). Thep55 GAG protein consists of different parts, which are processed by HIVprotease upon maturation of the particle into a functional infectiousparticle. The N-terminal matrix protein part ensures binding to themembrane via myristoylation and ensures budding (Bryant and Ratner,1990). The Capsid protein forms the cone-shaped viral core afterprocessing, while the nucleocapsid protein and the p6 protein bind toand protect the viral RNA. The p55 GAG protein is highly mobile beforeaccumulation in cholesterol-rich regions of the membrane, wheremultimerization actually initiates the budding process (Gomez and Hope,2006). A total of 4000-5000 GAG molecules are required to form a singleparticle with a size of about 145 nm (Briggs et al., 2004).

BRIEF SUMMARY

Surprisingly, it was found that the p55 GAG protein can be used to trapa bait protein together with its physiological binding partners intoVLPs that are budded from human cells. The very mild “extraction” or“abduction” of the protein complex ensures the identification ofrelevant interacting proteins. After introduction of a simple one-stepparticle enrichment protocol to speed up the workflow, it was found thatthis viral particle-based protein-protein interaction trap approach(called “Virotrap”) can be used for the detection of binaryinteractions. The identification of new partners by the coupling of theVirotrap process to MS-based analysis is also shown.

A first aspect of the disclosure is an artificial virus-like particle(called “Virotrap particle”), comprising (1) a viral particle-formingpolypeptide, (2) a first interaction polypeptide and (3) a secondinteraction polypeptide, interacting with the first interactingpolypeptide. As explained below, in one preferred embodiment, theviral-forming polypeptide and the first interacting polypeptide may betwo different polypeptide domains of a fusion protein (i.e., a fusionprotein consisting of at least two polypeptides derived from twodifferent proteins).

In another preferred embodiment, the viral particle-forming polypeptideand the first interacting polypeptide are independent proteins. Besidesthe first and the second interaction polypeptide, the virus-likeparticle may comprise other proteins, recruited to first and/or secondinteraction polypeptides, wherein all the proteins together form oneprotein complex. “Polypeptide” refers to a polymer of amino acids anddoes not refer to a specific length of the molecule. This term alsoincludes post-translational modifications of the polypeptide, such asglycosylation, phosphorylation and acetylation. A “virus-like particle,”as used herein, is a particle consisting at least of a viralparticle-forming protein, but preferably without the viral DNA or RNA.“Viral particle-forming proteins,” as used herein, are known to theperson skilled in the art and are proteins that allow the assembly ofviral particles and, preferably, budding of the particles of the cell.Examples of such particles have been described in the art and include,but are not limited to, particles derived from virus families includingParvoviridae (such as adeno-associated virus), Retroviridae (such asHIV), and Flaviviridae (such as Hepatitis C virus).

In a preferred embodiment, the particle-forming polypeptide may be amodification of the naturally occurring particle-forming protein, suchas a deletion and or mutation, as long as they do not inhibit theparticle formation. Preferably, the deletion and/or mutation is reducingthe binding of the particle-forming polypeptide with host proteins.Preferably, the modification is a fusion protein. Preferably, the viralparticle-forming polypeptide, or its modification, forms a viralstructure, consisting of a hollow particle, in which the first andsecond interaction polypeptides are trapped. In a preferred embodiment,the first interaction polypeptide is anchored to the viral structure,ensuring the capturing of the protein complex formed by the first andthe second interaction polypeptide into the inside of the virus-likeparticle. The anchoring may be direct, wherein the fusion partner of theviral particle-forming polypeptide acts as the first interactionpolypeptide, or indirect, wherein an independent linker molecule bindsto the viral particle-forming polypeptide at one hand, and to aconstruct comprising the first interaction polypeptide at the other hand(“dimerizing linker”) as illustrated in FIG. 1. As a non-limitingexample, such a linker may be a molecule as illustrated in FIG. 1, or itmay be a bispecific protein or peptide affinity ligand such as anantibody, or in a preferred setting, a bispecific NANOBODY® orALPHABODY® binding to the viral particle-forming polypeptide at onehand, and to the first interaction polypeptide at the other hand. Incase of a direct anchoring, the viral-forming polypeptide and the firstinteracting polypeptide are two different polypeptide domains of thesame protein. Preferably, the viral particle-forming polypeptide is aHIV protein; even more preferably, the viral particle-formingpolypeptide is the p55 GAG protein, or a modification or functionalfragment thereof. A “modification” or “functional fragment,” as usedherein, is a modification or functional fragment that is still capableof forming virus-like particles that are capable of entrapping theprotein complex according to the disclosure. Preferably, themodification is a fusion protein; even more preferably, the p55 GAGprotein is fused to the first interaction polypeptide.

Another aspect of the disclosure is the use of an artificial virus-likeparticle, according to the disclosure, for the detection ofprotein-protein interactions.

Still another aspect of the disclosure is a method for detectingprotein-protein interactions, the method comprising (1) the expressionof a viral particle-forming polypeptide in a cell, (2) recruiting afirst interaction polypeptide to the viral particle-forming polypeptide,(3) recruiting a second interacting polypeptide to the first interactionpolypeptide, (4) isolation of the virus-like particles, and (5) analysisof the entrapped protein complex. Preferably, the cell is a mammaliancell. Preferably, the analysis of the entrapped protein complex is anMS-based analysis. It is clear for the person skilled in the art thatprotein-protein interactions of any nature can be detected with themethod. As a non-limiting example, the method may be used to detectproteins involved in a signaling network, but it may also be used todetect antigen-antibody interactions, or other affinity-binding proteinsand their target(s).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Principle of conditional protein complex trapping in virotrapparticles. A bait protein is fused in frame to FKBP12. Upon addition ofMtx-PEG-FK506 dimerizer, the FKBP 12-bait fusion protein is recruited tothe GAG-eDHFR fusion protein, leading to trapping of the bait and itsinteracting proteins in the virotrap particles that are formed by GAGmultimerization and budding. Further purification of virotrap particlesfollowed by proteomic analysis results in identification of theinteracting proteins.

FIG. 2: Plasmid map for the pMET7-VT1-MCS vector. Transfer of the codingsequences for EGFP led to the pMET7-VT1-EGFP vector, respectively,transfer of the GATEWAY® cassette resulted in the pMET7-VT1-GW, whichthen allowed transfer of the CSK1B coding sequence from a GATEWAY® entryclone, leading to pMET7-VT1-CKS1B. MCS: multi-cloning site

FIG. 3: Western blot showing the interaction between CSK1B bait and CDK2prey upon addition of dimerizer (5 and 10 μM). HEK293T cells weretransfected with the pMET7-VT1-CSK1B or pMET7-VT1-EGFP bait constructs,combined with the E-tagged CDK2 prey construct. After purification, thenanoparticles were directly brought in 2×SDS-PAGE loading buffer andloaded for SDS-PAGE separation and Western blot analysis. The threeupper panels show samples prepared from purified particles. The lowerpanels are lysate samples obtained from the producer cells, confirmingsimilar expression levels for the different components.

FIG. 4: Panel A: Schematic representation of the Virotrap strategy.Expression of a GAG-bait fusion protein results in the submembranemultimerization and consequent budding of virotrap particles from thecells. Interaction partners of the bait protein are also trapped inthese virotrap particles and can be identified after purification and MSanalysis. Panel B: Supernatants from HEK293T cells transfected withdifferent combination of bait proteins (GAG-EGFP control and GAG-Ras)and FLAG-tagged prey proteins (IRS2 and RAF), were harvested after 24hours, and were processed by ultracentrifugation to pellet theparticles. Particle pellets were separated by SDS-PAGE and probed afterWestern blotting with anti-FLAG antibodies. Panel C: HEK293T cells wereseeded in six-well plates and transfected with bait and preycombinations as in Panel B. Both wild-type and E-tagged VSV-Gglycoproteins were expressed to allow particle enrichment via asingle-step protocol from a 1 ml harvest. Western blotting of the elutedparticles was performed with anti-FLAG and anti-GAG antibodies. Panel D:Virotrap experiments for two interaction pairs in both directions.Single-step purifications via anti-FLAG antibodies from six-welltransfections were loaded for Western blotting and probed withanti-Etag, anti-GAG and anti-Actin antibodies for both the enrichedparticles and the producer cell lysates. The expression of GAG-S100A1and GAG-S100B was below the detection limit in the lysates. Note thatexpression of the GAG protein without a fused bait does not lead todetectable particle formation.

FIG. 5: Detection of binary interactions with Virotrap. Comparison ofthe results for the positive (PRS) and the random reference set (RRS)obtained with the Virotrap system with other binary systems. Allinteractions from the PRS and RRS were explored by transfection insix-well plates, processing by a single-step purification protocol, andWestern blot analysis of the eluted particles and lysates of theproducer cells. The presence of prey proteins was revealed by anti-Etagantibodies on the VLP samples. The colored blocks show the Virotrapresults for 30% positive interactions at the expense of 5% falsepositive signals in the RRS set. For the other methods, data from Braunet al., 2009, was used.

FIG. 6: Identification of novel interaction partners using co-complex MSanalysis. Panel a: Analysis of the CDK2 interactome using Virotrap. Atotal of nine Virotrap experiments with on-bead VLP lysis was performed.The GAG-CDK2 identification list was challenged with identificationlists from mock and GAG-EGFP controls, from GAG-FADD and from fiveadditional Virotrap experiments. The CDK2 interactome (Right PanelTable) was obtained by removing all protein identifications found in theother experiments. *CKS1B was retained as it was also used as a GAG-BAITconstruct in the reference experiments. Note that the CDK2-CKS1Binteraction is a model interaction used for validation of the system.Panel b: The FADD interactome was obtained by adding three repeatexperiments and controls using a specific elution protocol (scheme onthe left). One of the FADD repeats was treated with TNFα duringproduction. The protein list (right panel table) was obtained byconsidering only confident identifications (at least two peptides) andby removing all proteins identified in the eleven other experiments. Thenumbers in brackets show the number of times the protein was identifiedin the four FADD experiments. *: interaction was shown with CaseinKinase 1 α(CSNK1A). **Affects FAS signaling Panel c: Confirmation of theA20-FADD interaction. A20 was found in two FADD Virotrap experiments.The interaction was confirmed by co-immunoprecipitation experimentsshowing specific binding of A20 to immune-precipitated FADD (leftpanels), or of FADD to immune-precipitated A20 (right panels). Taggedproteins (FLAG and VSV tags) were expressed in HEK293T cells and wereprecipitated after lysis using paramagnetic anti-FLAG beads. Theco-precipitated proteins were revealed by anti-VSV antibodies.

FIG. 7: Amino acid sequence of Desmoglein 1 (SEQ ID NO: 26) withannotation of extracellular, transmembrane and cytoplasmic regions, andwith mapping of the peptides identified in the Virotrap analysis withS100A1 bait protein.

DETAILED DESCRIPTION Examples Materials and Methods Plasmids andAntibodies

The p55 GAG fusion constructs were generated by PCR amplification usingprimers Oligo1 and Oligo2 (see Table 1) of the p55 GAG coding sequencefrom the pCMV-dR8.74 packaging construct (Addgene) and by subsequentIN-FUSION® reaction (Clontech) in pMG1-Ras, a Ras expression vector usedin the MAPPIT system (Eyckerman et al., 2001), resulting in a p55GAG-RAS under control of the strong SRalpha promoter (pMET7-GAG-Ras).EGFP was transferred from pEGFP-C1 vector (Clontech) to generate thepMET7-GAG-EGFP construct. Using PCR-based cloning, a GATEWAY® cassettewas inserted to allow recombination-assisted cloning. The complete setof positive and random reference clones were transferred in a singledirection (no bait-prey swap) using standard GATEWAY® cloning. Prey ORFsfrom these sets were transferred into a GATEWAY®-compatible pMET7expression vector with an N-terminal E-tag fused in frame.

The pMD2.g pseudotyping vector was kindly provided by D. Trono. ThepcDNA3-FLAG-VSV-G and pcDNA3-Etag-VSV-G are described elsewhere(Eyckerman et al., submitted).

Antibodies used for Western blot were anti-p24 GAG (Abcam), anti-FLAG(M2, Sigma Aldrich), anti-actin (Sigma Aldrich) and anti-E-tag (Phadia).Secondary antibodies were from LI-COR, and blots were digitally imagedusing an ODYSSEY® Imager system (LI-COR).

Cell Culture, Production and Purification of Virotrap Particles.

HEK293T cells were cultured in a humidified atmosphere at 8% CO₂ usinghigh-glucose DMEM (Invitrogen) complemented with 10% FCS andantibiotics.

Cells were transfected overnight the day after seeding with a standardcalcium phosphate transfection procedure. For ultracentrifugationexperiments, 25 μg of bait vector (GAG-EGFP and GAG-Ras) was transfectedand normalized to 50 μg with a mock vector, in 6×10⁶ cells seeded theday before in 75 cm² bottles. For concentration of the virotrapparticles, supernatant was harvested after 24 hours, centrifuged samplesfor 3 minutes at 1250×g to remove cellular debris and filtered thesupernatant through 0.45 mm filters. The samples were then centrifugedin a Beckman ultracentrifuge using a Ti41 swinging bucket rotor at 22000rpm. The supernatant was discarded and particle pellets werere-suspended directly in loading buffer for Western analysis.

For binary interaction assays, 650,000 HEK293T cells were seeded the daybefore transfection in six-well plates. On the day of transfection, aDNA mixture was prepared containing the following: 3.5 μg bait construct(pMET7-GAG-bait), 0.8 μg prey construct (pMET7-E-tag prey orpMET7-FLAG-Raf), 0.7 μg pMD2.G and 1.4 μg pcDNA3-FLAG-VSV-G. Followingovernight transfection, cells were washed once with PBS and 1 ml offresh growth medium was added to the wells. Cellular debris was removedfrom the harvested supernatant by 3 minutes centrifugation at 2000×g.The cleared medium was then incubated with 10 μl DYNABEADS® MyOne™Streptavidin T1 beads (Invitrogen) pre-loaded with 1 μg monoclonalANTI-FLAG® BioM2-Biotin, Clone M2 (Sigma-Aldrich®) according to themanufacturer's protocol. After 2 hours binding at 4° C. by end-over-endrotation, beads were washed two times with washing buffer (20 mM HEPESpH 7.4, 150 mM NaCl), and the captured particles were released directlyin 35 μl 2×SDS-PAGE loading buffer. A 5-minute incubation step at 65° C.before removal of the beads ensured complete release. After boiling, thesamples were loaded on a 10% SDS-PAGE gel, or on commercial 4-12%gradient gels (Biorad), and after separation, the proteins weretransferred to HYBOND®-C Extra nitrocellulose membranes (GE Healthcare).Lysates of the producer cells were prepared by direct addition of 200 μlRIPA buffer (50 mM TRIS.HCl pH 7.4, 150 mM NaCl, 1% NP40, 1% sodiumdeoxycholate, 0.1% SDS+Complete protease inhibitor cocktail [Roche]) tothe six-well plates after washing of the cells in chilled PBS. Thelysates were cleared by centrifugation at 13000×g, 4° C. for 15 minutesto remove the insoluble fraction.

The PRS and RRS were randomized and processed in sets of about 45 singlePPI measurements. Each set was loaded on two 4-12% gradient gels with 26slots (Biorad). Each set of measurements also contained the GAG-EGFPexpression control, a mock control and the interaction between GRAP2 andLCP2 as a positive control for Virotrap functionality. A single pooledpositive control for the GRAP2-LCP2 interaction was also loaded on eachgel to allow cross-comparison between the gels. Bands were quantified byfluorescence signals with an ODYSSEY® system (LICOR). The detectionthreshold was based on RRS signals and was determined for eachindividual gel.

For mass spectrometry, 4.75×10⁶ HEK293T cells seeded in 75 cm² bottleswere transfected the next day with a total of 50 μg DNA. The followingDNA quantities were used: GAG-bait 25 μg; mock vector 17.8 μg; 7.2 μg ofa 50/50 pMD2.G-pcDNA3-FLAG-VSV-G mix. The cellular supernatant washarvested after 32 hours and was centrifuged for 3 minutes at 450×g toremove cellular debris. The cleared supernatant was then filtered using0.45 μm filters (MILLIPORE®). A total of 100 μl MyOne™ Streptavidin T1beads pre-loaded with 10 μl ANTI-FLAG® BioM2-Biotin antibody was used tobind the tagged particles. Particles were allowed to bind for 2 hours byend-over-end rotation. Bead-particle complexes were washed once withwashing buffer and were then frozen overnight in lysis buffer (PBS, 0.4%CHAPS, 1.2 M guanidine hydrochloride) to release and denature thetrapped proteins. After lysis of the particles and removal of the beads,proteins were reduced (10 mM TCEP.HCl, 10 minutes at 37° C.) andalkylated (20 mM Iodoacetamide, 10 minutes at 37° C.). Via NAP5 gelfiltration columns (GE Healthcare), the protein sample was transferredto 10 mM ammonium bicarbonate buffer. Trypsin digest was performedovernight at 37° C. using 0.5 μg sequence-grade trypsin (Promega).Samples were vacuum dried and resuspended in 2% acetonitrile, separatedby nano-LC and directly analyzed with a LTQ®ORBITRAP® Velos instrument(Thermo Scientific). Searches were performed using the MASCOT® algorithmat 99% confidence against the human Swissprot database complemented withHIV-1 and EGFP protein sequences.

Example 1 Generation of the Conditional Trapping Construct

The p55 GAG fusion constructs were generated by PCR amplification usingprimers Oligo1 and Oligo2 (see Table 1) of the p55 GAG coding sequencefrom the pCMV-dR8.74 packaging construct (Addgene) and by subsequentIN-FUSION® reaction (Clontech) in a pMET7-gp130-RAS construct (Eyckermanet al., 2001). This resulted in a p55 GAG-fusion construct under controlof the strong SRalpha promoter. The plasmid was designatedpMET7-GAG-RAS. EGFP was transferred from pEGFP-C1 vector (Clontech) togenerate the pMET7-GAG-EGFP construct. The eDHFR fragment was amplifiedfrom plasmid pSEL1-eDHFR (Caligiuri et al., 2006) with primers Oligo3and Oligo4, digested with XhoI and XbaI and cloned in the SalI-XbaIopened pMET7-GAG-RAS backbone, which resulted in pMET7-GAG-eDHFR. TheFKBP12 protein was amplified with primers Oligo5 and Oligo6 frompMG2-FKBP12 (Eyckerman et al., 2005). The PCR product was digested withNdeI and XbaI and cloned in the NdeI-XbaI opened pMET7-GAG-eDHFR vector,which resulted in the pMET7-GAG-eDHFR-FKBP12-MCS construct. The reverseprimer Oligo6 also encoded a flexible Gly-Gly-Ser hinge sequence andcontained a number of restriction enzyme recognition sites. Thismulti-cloning site (MCS) allows different cloning strategies for theC-terminal fusion of a bait protein. The primers Oligo7 and Oligo8 wereannealed and ligated into the NdeI-MluI openedpMET7-GAG-eDHFR-FKBP12-MCS construct to insert a FLAG tag sequence and aT2A auto-processing site. This resulted inpMET7-GAG-eDHFR-T2A-FLAG-FKBP12-MCS. The Thosae asigna 2A (T2A)auto-processing sequence ensures, by a ribosomal skip mechanism(Szymczak et al., 2004), the complete cleavage of the fusion proteinresulting in two protein fragments upon translation: the GAG-eDHFR partand the FKBP12-MCS part. The EcoRI site that was present within theeDHFR coding sequence was removed by using site-directed mutagenesis(QUICKCHANGE™ Site-Directed Mutagenesis kit, Stratagene) with Oligo9 andOligo10 on pMET7-GAG-eDHFR-T2A-FLAG-FKBP12-MCS, resulting in thepMET7-VT1-MCS construct.

The GATEWAY® cassette (Invitrogen) was amplified by primers Oligo11 andOligo12 from pMG1-Gateway (Braun et al., 2009), and cloned via MfeI-XbaIin the EcoRI-XbaI opened pMET7-VT1-MCS plasmid, which resulted in thepMET7-VT1-GW destination vector. The coding sequence for CSK1B wastransferred via the GATEWAY® LR reaction from the Positive Reference Setdescribed in Braun et al. (Braun et al., 2009) into the pMET7-VT1-GWdestination vector resulting in pMET7-VT1-CSK1B. The coding sequence forCDK2 was transferred by the LR reaction to a pMET7-Etag-GATEWAYSconstruct (Lievens et al., unpublished) leading to pMET7-Etag-CDK2.

The pcDNA3-FLAG-VSV-G construct used for purification was generated asdescribed (Eyckerman et al., submitted). The pMD2.G construct expressingVSV-G under control of a strong CMV promoter was provided by DidierTrono (EPFL, Lausanne, Switzerland).

The chemical bivalent molecule or dimerizer consists out of methotrexate(Mtx) and FK506 linked via a PolyEthylene Glycol (PEG) linker, and wasprepared as described in Caligiuri et al., 2006.

TABLE 1 Oligonucleotides used for the generation of the pMET7-VT1 constructs. SEQ ID Number Sequence Use NO Oligo1CTCTAAAAGCTGCGGGGCCCGCTAGCGCC GAG amplification 1 ACCATGGGTGCGAGAGCGTCAGOligo2 TGTATTCGGTGAATTCTGAGCTCGTCGAC GAG amplification 2CCGCCTTGTGACGAGGGGTCGCTGC Oligo3 GCGACTCGAGCGGAATCAGTCTGATTGCG eDHFR 3 Gamplification Oligo4 CGCTTCTAGATTACATATGGCCGCTGCCC eDHFR 4CGCCGCTCCAGAATCTC amplification Oligo5 GCGACATATGGGCACGCGTGTGCAGGTGFKBP12 5 GAAACCATCTC amplification Oligo6 CGCTTCTAGATTACTCGAGTGCGGCCGCGFKBP12 6 AATTCTGAGCTCGTCGACCCGCCTTCCAG amplification TTTTAGAAGCTCCOligo7 TATGGAGGGCAGAGGCAGCCTGCTGACCT T2A and FLAG 7GCGGCGACGTGGAGGAAAACCCCGGCCC sequence annealingCGATTACAAGGATGACGACGATAAGA Oligo8 CGCGTCTTATCGTCGTCATCCTTGTAATCGT2A and FLAG 8 GGGCCGGGGTTTTCCTCCACGTCGCCGCA sequence annealingGGTCAGCAGGCTGCCTCTGCCCTCCA Oligo9 GAATCGGTATTCAGCGAGTTCCACGATGCEcoRI mutagenesis 9 TGATG Oligo10 CATCAGCATCGTGGAACTCGCTGAATACCEcoRI mutagenesis 10 GATTC Oligo11 CCCCAATTGACAAGTTTGTACAAAAAAGCGATEWAY ® 11 cassette amplification Oligo12 GGGTCTAGATCAAACCACTTTGTACAAG GATEWAY ® 12 cassette amplification

Example 2 Production, Harvest and Western Blot Analysis

For production of virotrap particles, a co-transfection in HEK293T cellswas performed via the Ca-Phosphate precipitation method. HEK293T cellswere cultured in DMEM medium (Gibco) and 10% FCS at 37° C. in ahumidified atmosphere with 5% CO₂. The day before transfection, 650,000cells were seeded in a six-well plate. On the day of transfection, a DNAmixture was prepared containing the following:

-   -   0.7 μg pMD2.G    -   1.4 μg pcDNA3-FLAG-VSV-G    -   0.8 μg pMET7-Etag-CDK2    -   3.5 μg pMET7-VT1-CKS1B or pMET7-VT1-EGFP    -   15 μl of 2.5 M CaCl₂    -   Water was added to a total volume of 150 μl.

The DNA mixture was then added dropwise to 150 μl 2×HeBs solution whilevortexing. The transfection mix was brought on the cells and theprecipitates were left overnight for transfection. Followingtransfection, the cells were washed once with PBS and 1 ml of freshgrowth medium was added with either 5 or 10 μM of dimerizer, or withoutdimerizer. The production medium was harvested after 24 hours. Cellulardebris was removed by one minute centrifugation at 2000×g. The clearedmedium was then incubated with 10 μl DYNABEADS® MyOne™ Streptavidin T1beads (Invitrogen) loaded with 1 μg monoclonal ANTI-FLAG® BioM2-Biotin,Clone M2 (Sigma-Aldrich®) according to the manufacturer's protocol.After 2 hours binding at 4° C. by end-over-end rotation, beads werewashed two times with washing buffer (20 mM HEPES pH 7.4, 150 mM NaCl),and the captured nanoparticles were released directly in 35 μl2×SDS-PAGE loading buffer. The samples were incubated for 5 minutes at65° C. to ensure complete denaturation/release of the virotrapparticles. After removal of the beads and boiling, the samples wereloaded on a 10% SDS-PAGE gel, and after separation, the proteins weretransferred to HYBOND®-C Extra nitrocellulose membranes (GE Healthcare).Lysates of the producer cells were prepared by direct addition of 200 μlRIPA buffer (50 mM TRIS.HCl pH 7.4, 150 mM NaCl, 1% NP40, 1% sodiumdeoxycholate, 0.1% SDS+Complete protease inhibitor cocktail [Roche]) tothe six-well plates after washing of the cells in chilled PBS. Thelysates were cleared by centrifugation at 13000×g, 4° C. for 15 minutesto remove the insoluble fraction. Western blots were probed with mouseanti-E tag (Phadia, 1/1000), mouse anti-GAG (Abcam, 1/1000) or rabbitanti-VSV-G (Sigma/Aldrich, 1/5000). Secondary antibodies were fromLI-COR, and blots were digitally imaged using an ODYSSEY® Imager system(LI-COR).

Example 3 The pMET7-VT1 Construct

First, a plasmid for expression of the bait construct was generated. Thep55 GAG fragment was cloned in the pMET7 vector, which drives expressionvia the strong SRα promoter. The E. coli-derived Dihydrofolate reductase(DHFR) protein was fused C-terminally of GAG while the FK506-BindingProtein 12 (FKBP12) coding sequence was cloned via a T2A auto-processingsite and a FLAG-tag, in frame and C-terminal of DHFR. TheGAG-eDHFR-T2A-FKBP12 expression construct was followed by amulti-cloning site to allow efficient transfer of bait proteins intothis expression vector (FIG. 2).

A GATEWAY® cassette and an EGFP expression construct were inserted inthe MCS of pMET7-VT1-MCS by standard cloning procedures. The CSK1Bprotein was transferred via the GATEWAY® LR reaction into thepMET7-VT1-GATEWAY® construct.

Example 4 Test of the CKS1B-CDK2 Interaction

A well-described protein-protein interaction pair was used to test theconditional trapping in nanoparticles. The coding sequence for the CDC28Protein Kinase Regulatory Subunit 1B (CKS1B) protein was fused to theFKBP12 in the VT1 vector. Addition of the chimeric dimerizer molecule,which consists of methotrexate (Mtx) and FK506 linked by a polyethyleneglycol linker, would thus result in the recruitment of the CKS1B baitprotein to the GAG protein and to the forming nanoparticles (FIG. 3).The pMET7-VT1-EGFP bait construct was also transfected as a control forirrelevant associations. The interaction partner Cyclin-Dependent Kinase2 (CDK2), which has an N-terminal E-tag sequence, was co-expressed withboth the CSK1B bait protein and the EGFP control protein. Three separatetransfections were performed for each bait construct in combination withthe CDK2 prey. The first series was left untreated to verifydimerizer-independent interactions, while the second and third serieswere treated with 5 and 10 μM of dimerizer, respectively. Afterenrichment and direct elution in SDS loading buffer, the samples wereloaded on a 10% PAGE gel and transferred to nitrocellulose membranesafter migration. The membranes were first probed with antibodiesdirected against the E-tag revealing presence of the prey protein indimerizer-treated and bait-specific conditions. Expression of GAG andVSV-G was verified by using specific antibodies. The expression of theprey (E-tag), GAG and VSV-G was also monitored in the lysates from theproducer cells to ensure equal protein levels.

Clear dimerizer-specific recruitment of the prey construct to theparticles can be shown in case of co-expression of the bait CSK1B andprey CDK2. Some weak background association independent from thedimerizer is observed when the EGFP bait is expressed.

Example 5 Evaluation of the Viral Particle Trap

To remove the homogenization step in classical AP-MS strategies, it wasreasoned that incorporation of a protein complex inside a secretedvesicle should “trap” the interactions under native conditions andshould protect the complex during the downstream purification process.As expression of the HIV 1 p55 GAG protein allowed the formation ofsecreted particles, the concept of packaged or “wrapped” proteincomplexes was explored by the generation of a plasmid for the expressionof GAG fused in frame to a bait protein. A flexible hinge sequence wasinserted to limit sterical interference. FIG. 4, Panel A shows aschematic presentation of the Virotrap concept. The N-terminus of GAG isessential for membrane association through myristoylation and shouldthus remain available (Bryant and Ratner, 1990). All domains requiredfor multimerization were still present in the expression construct. As afirst PPI pair to evaluate the concept, the H-Ras protein that lackedthe myristoylation signal as a bait was selected, combined with the cRAFprey protein. A GAG-EGFP construct and an IRS2 prey were used asirrelevant bait and prey, respectively. Both preys contained anN-terminal FLAG tag to facilitate detection. A first method of particleenrichment was ultracentrifugation, a well-described strategy for theconcentration of lentiviral particles for various cell biologicalapplications. After co-expression of bait and prey proteins, cellsupernatants were harvested, filtered and centrifuged. After removal ofthe supernatant, the pelleted particles were resuspended directly inloading buffer and loaded for SDS-PAGE. After Western blotting andrevelation of the tagged cRAF protein, clear enrichment of the preyprotein could be demonstrated only when the H-Ras bait protein waspresent (FIG. 4, Panels B and C). Expression controls for the particlesand the producer cell lysates showed comparable expression for all baitand prey constructs. These results showed that the interaction betweenH-Ras and cRAF can be detected by co-packaging of bait and prey insecreted particles from live cells. A “mock” empty vector for baitexpression was also employed to exclude enrichment of the prey inexosomes that were also pelleted by the ultracentrifugation procedure.

To remove the tedious ultracentrifugation step, a single-step enrichmentprotocol for particles in the supernatant was developed and optimized.Briefly, co-expression of the classical VSV-G pseudotyping construct,together with a tagged variant of this glycoprotein, resulted in optimalpresentation of the purification tag on the surface of the particles.Paramagnetic beads containing immune reagents for the affinity tag werethen employed to capture the particles from the supernatant. First, theinteraction between H-Ras and cRAF was confirmed using this newpurification strategy. In this case, the system was based on theco-expression of untagged VSV-G with E-tagged VSV-G for capture andpurification. HEK293T cells were transfected in a six-well format withGAG-RAS bait together with FLAG-RAF prey, and the controls used in theultracentrifugation experiment. Virotrap particles were produced for 24hours and were harvested in 1 ml of supernatant. After purificationusing anti-E antibodies coupled to paramagnetic particles, SDS-page andWestern blotting, the preys were revealed using ANTI-FLAG® antibodies.Clear and specific enrichment of cRAF-prey was shown for the H-Ras-baitprotein. No, or very little, cRAF was revealed in case of a-specificbait, while no detectable irrelevant IRS2-prey was found for theH-Ras-bait.

The interaction between two protein pairs in both directions was thenexplored. These protein interactions were selected based on literatureevidence on their confirmation in independent methods (Braun et al.,2009), and because both protein partners reside in the cytoplasm. Inthis case, the purification strategy with a FLAG-tagged VSV-G variantwas used to replace the E-tagged VSV-G glycoprotein in the previousexperiment. After purification by the one-step protocol using ANTI-FLAG®resin, direct elution in PAGE loading buffer, and Western blotting forthe E-tag, interactions between CDK2 and CKS1B and between S100A andS100B were readily detected. By swapping bait and prey, the interactionsin both directions were shown (FIG. 4, Panel D). As controls, irrelevantbait and prey constructs were used.

By design, the Virotrap system is ideally suited to study cytoplasmicinteractions. To explore the uses and limitations and to compare toother existing technologies, the concept was evaluated by testing thehuman positive reference set (hsPRS-v1). The positive reference setconsists of 92 PPIs that were selected based on literature data, whilethe random reference set is generated using 188 randomly selectedproteins (hsRRS-v1; (Venkatesan et al., 2009). Both sets containproteins from all cellular compartments to remove any bias inlocalization. All PPIs from the PRS were tested in the Virotraptechnology by recombination-assisted transfer of one bait set in fusionto the GAG protein. Prey ORFs were transferred to an expression vectorresulting in N-terminally E-tagged fusion proteins. The PPIs were testedin a single direction implying no swap of bait and prey constructs. Thesame strategy was used for the RRS set, again without swapping of baitand prey proteins. All experiments were performed by transfection ofbait and prey expression vectors in HEK293T cells in a six-well format.One day after transfection, supernatants were harvested and processedusing the one-step purification protocol. Enriched particles were elutedfrom the paramagnetic beads in PAGE loading buffer and loaded onSDS-PAGE gels. A total of about 184 binary virotrap experiments wereperformed. Apart from positive and negative controls, the experimentswere controlled for transfection and for immunoblotting efficiency. Thepresence of prey proteins in purified particles was revealed via anti-Etag immunoblots. The threshold of detection of true positives versusfalse positives was set for every individual gel. This led to thedetection of 28 (31%) interactions in the PRS, while five (5%)interactions were detected in the RRS. Expression of the bait proteinfusions in the lysates of the producer cells was verified, which showedthat approximately 30% (56 out of 184 bait fusions) was not expressed ata detectable level. Although this could be explained by structuralconstraints in the fusion proteins or by interference with particleformation, only detectable expression was observed for 25 out of 41tested prey proteins (61%) of the prey proteins in the producer lysates,where only an N-terminal E-tag was inserted before the protein.Therefore, it is believed that the current data provides anunderestimate of the detectable interactions. FIG. 5 shows the overlapbetween the Virotrap data and data obtained with other PPI methods forthe PRS and RRS as published by Braun and colleagues (2009). Seveninteractions out of the positive reference set can be detected with allmethods, while nine interactions are unique for the Virotrap method,proving the unique application window for the technology.

Example 6 Discovery of Novel Interaction Partners Using Co-ComplexVirotrap

For the detection of novel interaction partners, the purificationprocedure was scaled up to compensate for a larger production scale.

The protein complexes of two cytosolic proteins were investigated inmore detail: Cyclin-Dependent Kinase 2 (CDK2) and Fas-Associated viaDeath Domain (FADD). An important issue in typical MS co-complexstrategies relates to the background. The background in Virotrap willcontain GAG- and VSV-G-derived peptide sequences, together with hostbinding partners, proteins implicated in budding, and serum proteinsassociated with the outside of the VLPs. To define these backgroundproteins, additional experiments in the design of this study wereincluded to allow the construction of a comprehensive background list.In its simplest format, this list can be subtracted from the proteinidentifications that are specific for the bait (FIG. 6, Panel a). Atotal of nine experiments were performed (mock control, EGFP bait andfive additional bait constructs). Cells were co-transfected with baitproteins and constructs for purification. After harvest andpurification, particles were lysed directly on the beads using achaotropic lysis buffer. The lysates were then processed by reductionand alkylation, buffer exchange and trypsin digest. MS analysis followedby identification via MASCOT (99% confidence) resulted in theidentification of between 140 (for LCP2 bait) to 277 (FADD) proteins byat least two unique peptides (Table 2). By comparing the identificationlists, a background list of 306 proteins that were found in at least twoof the lists were extracted for the different experiments (Table 3). Bysubtraction of this list from the CDK2 identification list, a limitedset of 15 putative binding partners remains. Further removal of proteinidentifications that were also found with a single peptide in otherexperiments revealed a short list of seven binding partners. For four ofthese candidates, there is clear evidence in literature (FIG. 6, Panela). The list of unique proteins for FADD is more extensive (73proteins), even after removal of proteins that were additionally foundin one of the other experiments with a single peptide (35 proteins,Table 4). It is clear that this list contains real binding partners(Receptor interacting protein 1 RIPK1, Casein Kinase 1 alpha andepsilon) as well as unlikely binders. Therefore, the analysis of FADDusing additional Virotrap experiments was extended by using a specificelution protocol. In these experiments, the particles from theFLAG-antibody beads were eluted by competition with FLAG-peptide. Theparticles were then lysed in SDS, processed with detergent removalcolumns, and digested by trypsin. Controls included in these experimentswere mock, EGFP bait and an expression construct of a GAG variantwithout a bait. Three additional experiments for the FADD interactomewere performed (FIG. 6, Panel b, Table 5 for overview). Combination ofthe identifications from these experiments and the previous nineVirotrap assays resulted in a specific list of three candidate partnersidentified uniquely with at least two peptides in all four FADDexperiments: Syndecan 4 (SDC4), Casein kinase 1 epsilon (CSNK1E) andRIPK1. The list of candidate partners identified in two out of fourexperiments also contained two additional kinases: YES1 andCyclin-Dependent Kinase 1 (CDK1) (FIG. 6, Panel b, right side table).

Relaxing the criteria where all identifications (including singlepeptide identifications) in fewer of the repeat samples were included,revealed FAS receptor as candidate interaction partner in the lists ofidentifications, while TRADD was also found in a single FADD experiment.A20 (TNFAIP3) was identified in the first experiment series, and couldbe confirmed upon treatment of the cells with TNFα during Virotrapparticle production. The interaction between FADD and A20 was also shownby orthogonal co-immunoprecipitation (Co-IP) experiments (FIG. 6, Panelc).

TABLE 2 Overview of the proteomics data obtained for different GAG-baitconstructs. Results were obtained by searches of MS/MS data against allhuman and bovine SWISSPROT accessions, complemented with HIV-1, EGFP andVSV-G sequences by using MASCOT ® software. False discovery rates (FDRs)were determined by MASCOT ® searches against a database containing allsequences after reversion. Numbers are shown for all identified proteins(all) or proteins identified with at least two peptides (>1 peptide).Unique proteins were obtained by only considering proteinidentifications with at least two peptides after removal of proteinsthat were identified in one of the other Virotrap experiments.

TABLE 3 List of background proteins that were found in at least two outof nine Virotrap experiments. Both SWISSPROT accessions and gene namesare shown, as well as the number of Virotrap experiments containing theprotein (x/9). Times accession Gene symbol identified A5A3E0 POTEF 9A5PJE3 FGA 9 E1B7N2 LOC619094 9 E1B8G9 HIST3H2BB 9 E1B953 TUBB 9 E1B9F6LOC100848359 9 E1B9K1 UBC 9 E1BH06 LOC617696 9 E1BJB1 TUBB2A 9 F1MI18LOC506828 9 F1MNF8 LOC100141266 9 F1MNW4 ITIH2 9 F1MRD0 ACTB 9 F1MSZ6SERPINC1 9 F1MYN5 FBLN1 9 F1N5M2 GC 9 G3N2V5 HSP90AB1 9 G3X6N3 TF 9G5E513 Bt.12809 9 O00560 SDCBP 9 O46375 TTR 9 P00735 F2 9 P01966 HBA 9P02081 HBBF_BOVIN Hemoglobin fetal subunit beta 9 OS = Bos taurus PE = 1SV = 1 P02769 ALB 9 P07437 TUBB 9 P07900 HSP90AA1 9 P08107 HSPA1A 9P08670 VIM 9 P11142 HSPA8 9 P12268 IMPDH2 9 P12763 AHSG 9 P15497 APOA1 9P22626 HNRNPA2B1 9 P28800 SERPINF2 9 P34955 SERPINA1 9 P35580 MYH10 9P56652 ITIH3 9 P81187 CFB 9 P81644 APOA2 9 Q00839 HNRNPU 9 Q03247 APOE 9Q07020 RPL18 9 Q08431 MFGE8 9 Q13813 SPTAN1 9 Q3SZ57 AFP 9 Q3SZR3 ORM1 9Q3ZBS7 VTN 9 Q58D62 FETUB 9 Q7SIH1 A2M 9 Q9BQA1 WDR77 9 A2I7M9SERPINA3-2 8 F1MGU7 FGG 8 F1MMK9 AMBP 8 Q29443 TF 8 Q2KJF1 A1BG 8 Q3SZV7HPX 8 F1MBQ8 DDX5 8 F2Z4C1 TUBA3C 8 G5E507 HSP90AB1 8 P17697 CLU 8P35527 KRT9 8 P60709 ACTB 8 Q3SYW6 EIF3C 8 A1A4R1 HIST2H2AC 8 P01045KNG2 8 P26373 RPL13 8 12831136 gb|AAK08483.1|AF324493_2 gag polyprotein8 [HIV-1 vector pNL4-3] A6NKZ8 YI016_HUMAN Putative tubulin betachain-like 8 protein ENSP00000290377 OS = Homo sapiens PE = 5 SV = 2Q28107 F5 8 P04264 KRT1 8 P13645 KRT10 8 A6QLG5 RPS9 8 A7E350 PLG 8E1BEG2 HNRNPA3 8 G8JKY0 RPS8 8 P15311 EZR 8 P39023 RPL3 8 P62424 RPL7A 8A2I7N3 SERPINA3-7 7 A7E307 DDX17 7 F1MJH1 GSN 7 F1MY44 HNRNPM 7 G1K122RBP4 7 O43175 PHGDH 7 P00003 FLAG-VSVG 7 P12259 F5 7 P61978 HNRNPK 7Q9TTJ5 RGN 7 Q3Y5Z3 ADIPOQ 7 E1BF20 HNRNPH1 7 G5E5T5 G5E5T5_BOVINUncharacterized protein 7 (Fragment) OS = Bos taurus PE = 4 SV = 1296556483 gb|AAK08484.2|AF324493_3 pol polyprotein 7 [HIV-1 vectorpNL4-3] E1B7J1 E1B7J1_BOVIN Elongation factor 1-alpha 7 OS = Bos taurusPE = 3 SV = 1 E1BAK6 DAZAP1 7 F1MWU9 HSPA6 7 P40429 RPL13A 7 A5D9B4HNRPH2 7 E1BHA5 E1BHA5_BOVIN Uncharacterized protein 7 OS = Bos taurusPE = 4 SV = 1 P09651 HNRNPA1 7 P11940 PABPC1 7 P61353 RPL27 7 A2VE06RPS4Y1 7 G3N262 G3N262 BOVIN Uncharacterized protein 7 OS = Bos taurusPE = 3 SV = 1 P62269 RPS18 7 P26038 MSN 7 A7YW45 PRMT5 6 G3MYZ3 AFM 6Q05443 LUM 6 F1MY85 C5 6 A6NHL2 TUBAL3 6 P60842 EIF4A1 6 F1MQ37 MYH9 6A6QPP2 SERPIND1 6 P35579 MYH9 6 P35908 KRT2 6 P29966 MARCKS 6 E1B7R4EIF3A 6 P13639 EEF2 6 P12277 CKB 6 P23528 CFL1 6 E1B8G4 E1B8G4_BOVINUncharacterized protein 6 OS = Bos taurus PE = 3 SV = 2 P62917 RPL8 6Q8IX12 CCAR1 6 P04406 GAPDH 6 P14618 PKM 6 Q0VCZ3 YTHDF2 6 G3MX91 TARDBP6 F1MI47 RBM14 5 A5D784 CPNE8 5 G3N0S9 LOC515150 5 F1MNV5 KNG1 5 Q2UVX4C3 5 A5PK20 HIST1H1E 5 F1MYC9 SPTBN1 5 F1MVC0 CAD 5 E1BGR6 E1BGR6_BOVINUncharacterized protein 5 OS = Bos taurus PE = 3 SV = 1 P62249 RPS16 5P83731 RPL24 5 G3X861 G3X861_BOVIN Uncharacterized protein 5 (Fragment)OS = Bos taurus PE = 3 SV = 1 P62280 RPS11 5 P08865 RPSA 5 A5PKD6 GNB4 5F1MKC4 F1MKC4_BOVIN Uncharacterized protein 5 OS = Bos taurus PE = 3 SV= 2 P26641 EEF1G 5 Q06830 PRDX1 5 P09543 CNP 5 F1MMD7 ITIH4 5 Q3T052ITIH4 4 P02768 ALB 4 P55884 EIF3B 4 P02538 KRT6A 4 A7MAZ5 HIST1H1D 4P35613 BSG 4 Q3SZH5 AGT 4 P39060 COL18A1 4 P60033 CD81 4 P62937 PPIA 4Q01082 SPTBN1 4 Q08E32 CHMP4B 4 A5PK61 H3F3C 4 Q13151 HNRNPA0 4 F1MB60RPS26 4 P23396 RPS3 4 F1MMP5 ITIH1 4 O15372 EIF3H 4 P46777 RPL5 4 Q02543RPL18A 4 G3X7A5 C3 4 E1BE42 E1BE42_BOVIN Uncharacterized protein 4 OS =Bos taurus PE = 3 SV = 1 Q9Y265 RUVBL1 4 O18789 RPS2 4 G3N2F0G3N2F0_BOVIN Elongation factor 1-alpha 4 OS = Bos taurus PE = 3 SV = 1P62913 RPL11 4 A6NMY6 ANXA2P2 4 E1BB17 HNRNPH3 4 P08779 KRT16 4 F6QVC9ANXA5 4 E1BNB4 PABPC1L 4 A6H769 RPS7 4 E1BAT6 E1BAT6_BOVINUncharacterized protein 4 OS = Bos taurus PE = 3 SV = 1 Q562R1 ACTBL2 4Q8WUM4 PDCD6IP 4 P19338 NCL 3 Q3SYR0 SERPINA7 3 P01614 KV201_HUMAN Igkappa chain V-II region 3 Cum OS = Homo sapiens PE = 1 SV = 1 P17690APOH 3 G3N361 NONO 3 P63243 GNB2L1 3 P84103 SRSF3 3 Q01130 SRSF2 3G8JKV5 RPL14 3 F1MJM0 ZNF326 3 F1ML72 RPL34 3 FlMSD2 RUVBL2 3 O15371EIF3D 3 P08621 SNRNP70 3 P18621 RPL17 3 G3X8B1 LOC613401 3 P62935 PPIA 3O43242 PSMD3 3 P41252 IARS 3 P49327 FASN 3 Q3MHL4 AHCY 3 Q86YQ8 CPNE8 3G5E604 G5E604_BOVIN Uncharacterized protein 3 (Fragment) OS = Bos taurusPE = 4 SV = 1 Q12906 ILF3 3 A7MB16 EIF3B 3 F1MH40 Bt.57604 3 E1BCL3LOC507211 3 P54727 RAD23B 3 G3N2D7 IGLL1 3 A7MBG8 RUVBL1 3 F1MZ00 SNRPD33 F1N6C0 F1N6C0_BOVIN Uncharacterized protein 3 OS = Bos taurus PE-4 SV= 2 Q13310 PABPC4 3 F1MXE4 PSMD6 3 A4IFP7 ARF5 3 F1N0E5 CCT4 3 P46779RPL28 3 A5PK39 TPP2 3 P78371 CCT2 3 P02786 TFRC 3 F1MPU0 CLTC 3 O14744PRMT5 3 Q969P0 IGSF8 3 Q9P2B2 PTGFRN 3 P07224 PROS1 2 Q28085 CFH 2F1MG05 EEF1G 2 F1MLW8 LOC100847119 2 E1BMJ0 LOC100847889 2 A0JND2 KRT802 B8Y9S9 FN1 2 P02656 APOC3 2 Q3MHN2 C9 2 F6QYV9 SSRP1 2 P02253 HIST1H1C2 P07910 HNRNPC 2 P08758 ANXA5 2 P43243 MATR3 2 Q9Y2W1 THRAP3 2 E1BQ37SFPQ 2 P11586 MTHFD1 2 A6QLT5 UBAP2L 2 E1B9M9 L00525863 2 P20645 M6PR 2Q96EP5 DAZAP1 2 P40227 CCT6A 2 P61024 CKS1B 2 G1K134 Bt.57435 2 P84090ERH 2 P30101 PDIA3 2 F1MZ92 YBX1 2 A4IFC3 PABPC4 2 A5PK63 RPS17 2 E1BCF5RPL26L1 2 F1MHJ6 F1MHJ6_BOVIN 60S ribosomal protein 2 L18a OS = Bostaurus PE = 3 SV = 2 F1MLH6 CALM2 2 P05543 SERPINA7 2 P13010 XRCC5 2Q15366 PCBP2 2 E1BF81 SERPINA6 2 P16403 HIST1H1C 2 G5E531 TCP1 2 P02788LTF 2 P08238 HSP90AB1 2 P62194 PSMC5 2 P04350 TUBB4A 2 P02533 KRT14 2Q5D862 FLG2 2 D3IVZ2 DDX3Y 2 075131 CPNE3 2 P57721 PCBP3 2 Q5VW32 BROX 2E1BKM4 PDCD6IP 2 P60660 MYL6 2 F1MZV2 CHMP5 2 O75340 PDCD6 2 P29144 TPP22 A5D9H5 HNRPD 2 A6NIZ1 RP1BL_HUMAN Ras-related protein Rap-1b- 2 likeprotein OS = Homo sapiens PE = 2 SV = 1 A7Z057 YWHAG 2 E1B726 PLG 2E1B7T4 E1B7T4_BOVIN Uncharacterized protein 2 OS = Bos taurus PE = 3 SV= 2 E1BK63 E1BK63_BOVIN Ribosomal protein L15 2 OS = Bos taurus PE = 3SV = 1 E1BNR0 Bt.110587 2 G3MYE2 G3MYE2_BOVIN Uncharacterized protein 2(Fragment) OS = Bos taurus PE = 3 SV = 1 O14828 SCAMP3 2 P00004 VSVG 2P05023 ATP1A1 2 P06733 ENO1 2 P08195 SLC3A2 2 P18124 RPL7 2 P27635 RPL102 P36578 RPL4 2 P49006 MARCKSL1 2 P52272 HNRNPM 2 P53985 SLC16A1 2P61204 ARF3 2 P62258 YWHAE 2 P62847 RPS24 2 Q02878 RPL6 2 Q14152 EIF3A 2Q15758 SLC1A5 2 Q53EZ4 CEP55 2

TABLE 4 List of FADD interaction partners. List of FADD interactionpartners after removal of all proteins (including single peptide proteinidentifications) that were found in at least one of the other Virotrapexperiments. Proteins in bold have been linked to FADD or to FASsignaling before. Accession Protein 1 A2VDY3 CHMP4A 2 A4FUC2 HNRNPUL1 3A6QLS9 RAB10 4 E1BAF6 PRRC2A 5 F1MND1 CDC42 6 F1MSI2 AGRN 7 F1MX61 SF3B18 G3N3Q3 G3N3Q3_BOVIN 9 G5E5V7 G5E5V7_BOVIN 10 O00232 PSMD12 11 O00299CLIC1 12 O60884 DNAJA2 13 P01891 HLA-A 14 P05556 ITGB1 15 P06493 CDK1 16P07195 LDHB 17 P11017 GNB2 18 P21580 TNFAIP3 19 P31431 SDC4 20 P31689DNAJA1 21 P48643 CCT5 22 P48729 CSNK1A1 23 P49674 CSNK1E 24 P60174 TPI125 P61247 RPS3A 26 P62871 GNB1 27 P62888 RPL30 28 Q13158 FADD 29 Q13546RIPK1 30 Q148F1 CFL2 31 Q8TB73 NDNF 32 Q99661 KIF2C 33 Q9NRX5 SERINC1 34Q9UN37 VPS4A 35 Q9Y5K6 CD2AP

TABLE 5 Overview of the proteomics data obtained for different GAG-baitconstructs using specific elution of particles from the purificationbeads. # Unique PRIDE # # # Proteins proteins Experiment # PeptideProteins (>1 (>1 FDR Accession Spectra sequences (all) peptide) peptide)(%) mock 28963 6706 557 169 95 14 0.6 sGAG* 28965 7661 595 255 117 180.4 GAG- 28964 6450 397 242 114 29 1.7 EGFP GAG- 28962 7546 864 375 17420 0.7 FADD GAG- 28960 8377 517 166 98 2 0.4 FADD GAG- 28961 8388 717231 121 9 0.5 FADD** Results were obtained by searches of MS dataagainst all human and bovine SWISSPROT accessions, complemented withHIV-1, EGFP and VSV-G sequences. False discovery rates (FDRs) weredetermined by MASCOT searches against a database containing allsequences after inversion. *an alternative codon-optimized GAG constructwithout a bait was used to generate particles. **GAG-FADD Virotrapparticles were produced in the presence of TNFα.

Example 7 Identification of Desmosomal Components

By employing a similar background removal strategy for the S100A1 baitas for the CDK2 bait (i.e., removal of all a-specific proteinsidentified) in the first set of nine experiments, a list of ten putativeinteraction partners identified with at least two peptides was obtained(Table 6). Remarkably, three components of the desmosome can be found inthis list (Desmoplakin DSP, Desmoglein 1 DSG1 and Junction PlakoglobinJUP), as well as two keratin proteins not found in other bait proteins(thus, not constituting classical contaminating keratins). Thesekeratins are known intermediate filament components that use thedesmosome for anchoring (Kitajima, 2013). The link between S100 proteinsand desmosomes is hinted in literature. The S100A10 and S100A11 can befound together with desmosomal proteins in the cornified envelope(Robinson et al., 1997). Various members of the S100 family of proteinshave been implicated in inflammatory skin disorders affecting theintegrity of the skin such as psoriasis (Eckert et al., 2004). Inaddition, it is clear that these calcium-binding proteins play animportant role in metastasis of tumors, both in the primary tumor cellsand the metastatic niche (Lukanidin and Sleeman, 2012). The S100A1protein also plays an important role in striated muscle and has beenimplicated in myocardial (dys)function and heart failure (Krause et al.,2009).

FIG. 7 shows the mapping of the identified peptides of Desmoglein 1 onthe amino acid sequence. Peptides from both the extracellular part andthe intracellular part of the protein were identified. In Table 7, theidentified peptides for Desmoglein 1 are shown with their MASCOT scores.This data clearly supports the fact that Virotrap allows the detectionof transmembrane prey proteins.

TABLE 6 Putative interaction partners for the S100A1 bait protein.Desmosomal or intermediate filament proteins are annotated in thecomments column. Accession Protein Comment P05089 ARG1 P13647 KRT5Intermediate filament P14923 JUP Desmosome component P15924 DSPDesmosome component P23297 S100A1 BAIT Q02413 DSG1 Desmosome componentQ6UWP8 SBSN Q8N1N4 KRT78 Intermediary filament Q96P63 SERPINB12 Q99816TSG101 Q9UK41 VPS28

TABLE 7 the identified peptides for Desmoglein 1 (DSG1, Swissprot Acces-sion Q02413) are shown with their respective MASCOT ® scores. start aaend aa MASCOT ® SEQ ID Peptide position positionmodified peptide sequence score NO I 439 445 NH2-TGKLTLK-COOH 33 13 E916 925 NH2-ESSNVVVTER-COOH 77 14 J1 326 332 NH2-TNVGILK-COOH 41 15 F198 213 NH2- 57 16 IIRQEPSDSPM(oxid)FIINR- COOH A 129 144NH2-ALNSMGQDLERPLELR- 48 17 COOH M + K 391 422 NH2- 84 18TYVVTGNMGSNDKVGDFVAT DLDTGRPSTTVR-COOH F 198 213 NH2-IIRQEPSDSPMFIINR-74 19 COOH D 220 238 NH2- 73 20 TM(oxid)NNFLDREQYGQYAL AVR-COOH L 423438 NH2-YVMGNNPADLLAVDSR- 107 21 COOH D 220 238 NH2- 75 22TMNNFLDREQYGQYALAVR- COOH J2 333 352 NH2- 49 23 VVKPLDYEAMQSLQLSIGVR-COOH G 87 105 NH2-ISGVGIDQPPYGIFVINQK- 118 24 COOH B 369 390 NH2- 86 25ASAISVTVLNVIEGPVFRPGSK- COOH

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1. An artificial virus-like particle comprising: a viralparticle-forming polypeptide; a first interaction polypeptide; and asecond interaction polypeptide that interacts with the first interactingpolypeptide.
 2. The artificial virus-like particle according to claim 1,wherein the viral particle-forming polypeptide is a fusion protein. 3.The artificial virus-like particle according to claim 1, wherein theviral particle-forming polypeptide is a fusion of HIV p55 GAG protein.4. The artificial virus-like particle of claim 1, wherein the firstinteraction polypeptide is anchored to the viral particle-formingpolypeptide.
 5. A method of detecting a protein-protein interaction, themethod comprising: utilizing the artificial virus-like particle of claim1 to detect the protein-protein interaction.
 6. A method for detecting aprotein-protein interaction, the method comprising: expressing a viralparticle-forming polypeptide in a cell; recruiting a first interactionpolypeptide to said viral particle-forming polypeptide; recruiting asecond interacting polypeptide to the first interaction polypeptide;isolating the virus-like particles; and analyzing the entrapped proteincomplex so as to detect the protein-protein interaction.
 7. Theartificial virus-like particle of claim 2, wherein the viralparticle-forming polypeptide is a fusion of HIV p55 GAG protein.
 8. Theartificial virus-like particle of claim 2, wherein the first interactionpolypeptide is anchored to the viral particle-forming polypeptide. 9.The artificial virus-like particle of claim 3, wherein the firstinteraction polypeptide is anchored to the viral particle-formingpolypeptide.
 10. The artificial virus-like particle of claim 7, whereinthe first interaction polypeptide is anchored to the viralparticle-forming polypeptide.
 11. A method of detecting aprotein-interaction, the method comprising: utilizing the artificialvirus-like particle of claim 2 to detect the protein-proteininteraction.
 12. A method of detecting a protein-interaction, the methodcomprising: utilizing the artificial virus-like particle of claim 3 todetect the protein-protein interaction.
 13. A method of detecting aprotein-interaction, the method comprising: utilizing the artificialvirus-like particle of claim 7 to detect the protein-proteininteraction.
 14. A method of detecting a protein-interaction, the methodcomprising: utilizing the artificial virus-like particle of claim 8 todetect the protein-protein interaction.
 15. A method of detecting aprotein-interaction, the method comprising: utilizing the artificialvirus-like particle of claim 9 to detect the protein-proteininteraction.
 16. A method of detecting a protein-interaction, the methodcomprising: utilizing the artificial virus-like particle of claim 10 todetect the protein-protein interaction.
 17. A method of detecting aprotein-interaction, the method comprising: utilizing the artificialvirus-like particle of claim 11 to detect the protein-proteininteraction.
 18. An artificial virus-like particle comprising: a viralparticle-forming polypeptide, which is a fusion protein comprising HIVp55 GAG protein; a first interaction polypeptide that is anchored to theviral particle-forming polypeptide; and a second interaction polypeptidethat interacts with the first interacting polypeptide.
 19. A method ofdetecting a protein-protein interaction, the method comprising:utilizing the artificial virus-like particle of claim 18 to detect theprotein-protein interaction.